Schistosomiasis is among the most common human parasitic diseases in the world. An estimated 207 million people are infected, 97% of whom live on the African continent. Praziquantel (PZQ) is the least expensive, easiest to use and most readily available of all current anti-schistosomal drugs. One problem associated with PZQ treatment is that it does not kill schistosomes for a period of 2-4 weeks after they infect the host. A second potential problem is the presence of drug resistance traits in natural populations of worms. As yet, neither the molecule to which PZQ binds nor its mechanism of action have been identified. Here, we propose to employ two complementary approaches to identify the schistosome molecule to which PZQ binds. We will exploit the fact that different life cycle stages of Schistosoma mansoni have differing susceptibilities to PZQ by employing microarrays to analyze the transcriptomes of miracidia, cercariae and mature schistosomes which are each PZQ sensitive and compare them to those of mother sporocysts and juvenile schistosomes which are relatively insensitive. Previous experience suggests that this should provide a small pool of candidate target genes for further study and may also identify downstream components of the biochemical pathway driven by the PZQ target. We also propose to identify the molecular target of PZQ by using a chemically engineered PZQ probe containing a diazirine group to covalently cross link the drug to its target and an alkyne group to which a reporter tag can be attached using click chemistry. Probe bound target will then be identified by chemiluminescent detection. We will confirm the correct target or biochemical pathway has been identified by 'knocking down'candidate gene expression using RNAi in mature schistosomes which should abolish the PZQ sensitive phenotype. Small molecule libraries of PZQ analogs will be used to define the pharmacophore of the molecular target of PZQ. Finally, we will investigate the molecular basis of variable sensitivity to PZQ in field isolates of S. mansoni. A genetically diverse population of S. mansoni derived from natural infections in Kenya will be selected in mice for their varied sensitivities to PZQ. The transcriptomes of these divergent populations will be compared using microarrays in an effort to determine if variable sensitivity is based on mutations to the PZQ target gene or differences in the expression of either (i) the target, (ii) a downstream component of the biochemical pathway driven by the PZQ target or (iii) a PZQ detoxification/clearance mechanism. More explicit knowledge of the binding target of PZQ and the mechanism of action of the drug will help us to devise much- needed improved assays for monitoring the emergence of resistance. PUBLIC HEALTH RELEVANCE: Schistosomiasis infects an estimated 207 million people, mostly in tropical Africa. There is only one available drug, praziquantel, that combats all forms of schistosomiasis but we do not yet know how this drug works. This project seeks to better understand the mechanism of action of praziquantel, thus facilitating the development of the next generation of therapeutics.